There is an increasing need for safe, long-term preventive treatments for non-melanoma skin cancer (NMSC), which is a particular threat to high risk groups such as the growing populations of immunocompromised solid organ transplant patients, HIV patients, and elderly non-Hispanic Caucasians. We have shown that a combination of the well-characterized chemopreventive vitamin A metabolite, all-trans retinoic acid (ATRA), with the bioactive dietary agent auraptene (AUR), can synergistically or supra-additively suppress skin squamous cell carcinoma (SCC) tumor growth in a mouse xenograft model. A long-term goal of our research is to develop effective chemopreventive treatments for epithelial cancers. In keeping with this goal, the primary aim of this project is to gain a better understanding of the effects of ATRA, and AUR on the development of skin SCC. Our overall hypothesis is that the ATRA + AUR combination is more effective than either agent alone due to the ability of ATRA to suppress Stat3 signaling, and AUR to suppress the NF-:B pathway and other pathways. Stat3 is a transcription factor that controls cell proliferation and survival, and is important in the regulation of apoptosis, proliferation, motility, and angiogenesis, all hallmarks of malignancy. Our laboratories and others have shown that Stat3 activity is constitutive in several malignant cell types and is required for initiation, promotion and progression to a more malignant phenotype in skin SCC. NF-:B is a survival signaling transcription factor also involved in the malignant phenotype of many cancers, including SCCs. Our preliminary results show that the ATRA+AUR combination suppresses the growth of tumors derived from the human skin SCC cell line SRB12-p9 (P9 WT), in the severe combined immunodeficient (SCID) mouse orthotopic tumorigenesis model. We will first examine the effects of a wider range of doses of ATRA 1 AUR against tumor volume in SCID mice. We will compare the effects of AUR treatment in the context of pharmacological suppression of Stat3 (ATRA treatment) to genetic suppression of Stat3, (stable expression of a dominant negative acting form of Stat3; S3DN cells). Tumors will be analyzed in detail for malignant properties and for suppression of Stat3 and NF-:B pathways. We will further verify our hypothesis by genetically blocking the Stat3 pathway (S3DN cells) and the NF-:B pathway (shRNA mediated knockdown of NF-:B), alone and in combination, in the P9WT SCC cells. The tumorigenic properties of the resulting pathway suppressed cells will be determined.